Post on 01-Mar-2020
Wattakaka volubilis leaf Extract Regulates the Oxidative
Status and Antioxidant Gene Transcripts in aluminium
sulphate Induced Hepatotoxicity
S. Usharani, R.Abinaya, M.Arulmozhi, R.Devika, P.Priyanga
Department of Biochemistry, Idhaya College for Women- Sarugani.
Corresponding author :
S. Usharani, Department of Biochemistry, Idhaya College for Women- Sarugani.
Email: em.sam12@gmail.com
Abstract
The ability of Wattakaka volubilis to protect against aluminium sulphate induced
oxidative stress and hepatotoxicity was evaluated in male albino rats. (n=6) and the rats treated
with saline throughout the study period, served as control (group I). Rats were treated
aluminium sulphate (50mg/kg b.w.; i.p.) for a period of 14 days (group II). Group III rats were
treated with Aluminium sulphate+ MEWV (200 mg/kg/b.w) dissolved in normal saline (1ml/kg
b.wt) orally for 14days. Group IV rats treated Aluminiumsulphate + Treated with silymarin (25
mg/kg/b.w) dissolved in normal saline (1ml/kg b.wt) orally for 14days. Group V
Wattakakavolubilis(200 mg/kg/b.w) dissolved in normal saline (1ml/kg b.wt) orally for
14days.The levels of hepatic lipid peroxidation, antioxidants, and molecular biomarkers were
estimated twenty-four hours after the lastAluminium sulphate injection. Pretreatment with
wattakakavolubilis leaf extract significantly reduced aluminium sulphate -induced elevationof
malondialdehyde levels and nearly normalized levels of glutathione and activity of glutathione S-
transferase, glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) in the liver.
Wattakakavolubilis leaf extract also attenuatedAluminium sulphate -induced downregulation of
hepatic mRNA expression levels of MMP – 2 Results of DNA fragmentation support the ability of
W.volubilis leaf extract to ameliorate aluminium sulphate -induced liver toxicity. Taken together,
our results demonstrate that W.Volubilis leaf extractis rich in natural antioxidants and able to
attenuate Aluminium sulphate -induced hepatocellular injury, likelyby scavenging reactive free
radicals, boosting the endogenous antioxidant defense system,and overexpressing genes
encoding antioxidant enzymes.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1065
Keywords : Aluminium sulphate, Wattakaka volubilis, Antioxidant enzymes TNF – alpha,
SDS PAGE , Wattakakavolubilis.
Introduction
Reactive oxygen species (ROS), include free radicals such as superoxide (O2) hydroxyl
radicles (OH), Peroxyl radicle (ROO) as well as non-radical species such as hydrogen peroxide
(H2O2) [1]. Invivo, such species are securely coupled at their site of generation or are detoxified
by endogenous antioxtative defenses, so as to preserve optimal cellular function. In pathological
condition, however, the detoxifying mechanism are often inadequate as excessive quantities of
ROS are generated. This resulting pro-oxidant shift, a process known as oxidative stress can
result in the degradation of cellular components viz., DNA, carbohydrates, polyunsaturated lipids
and proteins or precipitate enzyme inactivation, irreversible cellular dysfunction and ultimately
cell death, if the pro-oxidant-antioxidant balance is not restored. Furthermore, ROS play a
cardinal role in the a etiology of numerous diseases [2]. In recent years, there is an increasing
interest in finding antioxidant phytochemicals, because they can inhibit the propagation of free
radical reactions, protect the human body from disease[3].
Polyphenols constitute a large group of naturally occurring substances in the plant
kingdom, which include the flavonoids. The plant phenolics are commonly present in fruits,
vegetables, leaves, nuts, seeds, barks, roots and in other plant parts. These substances have
considerable interest in the field of food chemistry, pharmacy and medicine due to wide range of
favorable biological effects including antioxidant properties. The antioxidant property of
phenolics is mainly due to their redox properties. They act as reducing agents (free radical
terminators) , hydrogen donars, singlet oxygen quenchers and metal chelators [4]. The alteration
of gene expression is the most fundamental and effective way for the cells to respond to
extracellular signals and changes in their environment (D’Angio and Finkelstein 2000). Cellular
response to oxidative stress includes changes in the pattern of gene expression through
regulatory factors. One of the superoxide-radical-sensitive and redox-sensitive transcription
factors is the nuclear factor-kappaB (NF-κB) which is required for the expression of many
cytokines involved in the pathogenesis of diverse injuries. TNF-α is responsible for hepatic
injury as well as for complications after liver transplantation.[5]. TNF-α effects are antagonized
by interleukin-10 (IL 10). It protects the liver against pro-inflammatory cytokines, at least in part
by counteracting their pro-apoptotic effects [6]. On the other hand, TNF-α has not only negative
effects on the liver, but it also plays an important role in liver regeneration [7]. MMP-2 and
MMP-9 over-expression has been reported in cardiac, lung [9] and brain ischemia-reperfusion
injury. [8] Similarly to TNF-α, MMP-9 was found to be an important mediator of liver
regeneration [9]. Since ischemia-reperfusion injury is an example of oxidative stress, it is
important to investigate the expression of MMP-2 andMMP-9 in a model of metal-induced
oxidative stress
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1066
Aluminium is the third most abundant element of the Earth’s crust, is a non essential and
toxic metal in humans [10]. Aluminium and its salts are commonly used in daily life, a
widespread use that was enhanced by the belief that it is non -toxic and is quickly excreted from
the body in the urine. However, this element has a negative impact on human health [11]. Due to
its abundance, every organism contains small quantities of aluminium,[12] and it can be found in
practically all of the tissues of mammals, including the brain, liver, heart, kidney, blood and
bones [13].Aluminium accumulation in the liver leads to cholestasis [14]. There has been
considerable debate as to whether chronic exposure to aluminium is involved in neuro-
degenerative disorders, such as Alzheimer’s disease [15] dialysis, Parkinson’s dementia, [16, 17]
and hepatotoxicity [18, 19]. The toxic effects of aluminium appear to be mediated, at least in
part, by free -radical generation [20, 21] The treatment commonly used in aluminium disorders is
desferrioxamine [22].
Wattakaka volubilis belongs to the family (Asclepiadaceae). The plants are distributed
along subtropical of Malaysia and India, South China, Taiwan and Srilanka. The flora is
important in the Indian traditional system of medicine and is utilized to treat several diseases.
[23]Wattakaka volubilis widely practiced in Indian traditional medicines and the leaf paste to
treat rheumatic pain, cough, fever and wicked cold [24]. Leaf paste is removed along with pepper
to treat dyspepsia [25]. Bark paste, mixed with hot milk is utilized internally for treating urinary
troubles [26] , and leaf powder is taken orally along with cow’s milk have antidiabetic activity
[27]. In present study the Wattakakavolubilis leaf extract regulates the oxidative status and
antioxidant gene transcripts in aluminium sulphate inducedhepatotoxicity.
MATERIALS AND METHODS
Collection & Authentication of Plant
The fresh leaves of Wattakakavolubilis were collected from Trichirapalli August, 2012.
The plant was compared with voucher specimen (voucher specimen No. 001) deposited
andidentified by Dr. John Britto Rapinet Herbanium. St. Joseph College, Trichy. The leaves were
washed thoroughly with tap water, shade dried, homogenized to fine powder and stored in
airtight bottles.
Preparation of the extract
The leaves of Wattakakavolubilis were dried in shady condition and powdered. About200
gof powdered material was dissolved with 250 ml of methanol and theextract was prepared by
using soxhlet apparatus for about48hr. The extract was filtered and concentrated inrotary
evaporator at 35-40⁰C under reduced pressure and was stored in refrigerated condition for further
use.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1067
Drugs and chemicals
Aluminium sulphate and silymarin were purchased from sigma-aldrich chemical
company .The diagnostic kits required for enzymatic assays were purchased from Span
Diagnostics, India
Maintenance of animals
Adult male Albino Wister rats weighing 150-200 g were used for the present
investigation. They were housed in a clean polypropylene cage and maintained under standard
laboratory conditions (temperature 25±2⁰C with dark/light cycle 12/12h).They were fed with
standard pellet diet (Hindustan lever, Kolkata, india) and water adlibitum. The animals were
acclimatized to laboratory conditions for one month prior to experiment. All procedures
described were reviewed and approved by IAEC NO: 02/005/2014.
Experimental Design
Animals were randomized and divided into six groups (n = 6)
Group I : Control rat (saline treated)
Group II : Aluminium sulphate (50mg/kg/day) control and received normal saline
(Domingo,1995)[12]
Group III : Methanol Extract of Wattakakavolubilis (200mg/kgb.w) + Aluminium
sulphate respectively daily for 14 days.
Group IV : Aluminium sulphate + Silymarin(25mg/kg b.w;p.o.)daily for 14 days.
Group V : Wattakaka volubilis (200 mg/kg/b.w) daily for 14 days.
Antioxidant Parameters
Lipid peroxidation assay
Lipid peroxidation (LPO) was measured following the method of Fraga etal., [28]. Acetic
acid 1.5mL (20%; pH 3.5), 1.5 of TBA (0.8%), and 0.2mL of sodium dodecyl sulfate (8.1%)
were added to 0.1ml of supernatant and heated at 100°C cooled for 60 min. The mixture was
cooled, and 5mL of n-butanol: pyridine (15 : 1) mixture and 1mL of distilled water were added
and vortexed vigorously. After centrifugation at 1200g for 10min, the organic layer was
separated and the absorbance was measured at 532nm using a spectrophotometer.
Malonyldialdehyde (MDA) is an end product of LPO, which reacts with TBA to form pink
chromogen–TBA reactive substance. It was calculated using a molar extinction coefficient of
1.56 X 105M-1 cm-1 and expressed as nanomoles of TBARS mg-1 of protein.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1068
Superoxide dismutase assay
Superoxide dismutase (SOD) activity was analyzed following the method described by
[29] . Assay mixture contained 0.1mL of supernatant, 1.2mL of sodium pyrophosphate buffer
(pH 8.3; 0.052M), 0.1mL of phenazine methosulfate (186 mM), 0.3mL of nitroblue tetrazolium
(300 mM), and 0.2mL of NADH (750 mM). Reaction was started by the addition of NADH.
After Incubation at 30o°C for 90s, the reaction was stopped by the addition of 0.1mL of glacial
acetic acid. Reaction mixture was stirred vigorously with 4.0mL of n-butanol. Colour intensity of
the chromogen in the butanol was measured spectrophotometrically at 560nm and the
concentration of SOD was expressed as Umg-1 of protein.
Reduced glutathione assay
Reduced glutathione (GSH) was measured according to the method of Ellman [30] .The
equal quantity of homogenate was mixed with 10% trichloroacetic acid and centrifuged to
separate the proteins. To 0.01 ml of this supernatant, 2ml of phosphate buffer (pH 8.4), 0.5 ml of
5’5-dithio, bis (2-nitrobenzoic acid) and 0.4ml double distilled water were added. The mixture
was vortexed and the absorbance to be read at 412nm within 15 min. The concentration of
glutathione was expressed as μ g/mg of protein.
Catalase assay
Catalase activity (CAT) was measured following the method of Sinha [31] . A 0.1mL of
supernatant was added to the cuvette containing 1.9mL of 50mM phosphate buffer (pH 7.0).
Reaction was started by the addition of 1.0mL of freshly prepared 30mM H2O2. The rate of the
decomposition of H2O2 was measured spectrophotometrically at 240 nm. Activity of CAT was
expressed as Umg-1 of protein.
Glutathione peroxidase assay
Glutathione peroxidase (GPx) activity was determined following the method [32]. The
reaction mixture consist of 400μL of 0.25M potassium phosphate buffer (pH- 7.0), 200 mL
supernatant, 100 μL GSH (10 mM), 100 μL NADPH (2.5mM), and 100μL GRD (6UmL-1).
Reaction was started by adding 100μL hydrogen peroxide (12mM) and absorbance was
measured at 366nm at 1min intervals for 5 min using a molar extinction coefficient of 6.22X
103M-1cm-1. Data are expressed as mU mg-1 of protein.
Immunohistochemistry of TNF-alpha
Tumor tissue had been routinely 10% formalin fixed (24–72 hr) and paraffin embedded.
Four-µm-thick sections were cut using microtome and dried for 60 min at 65C. Sections were
dewaxed in Xylene Substitute and rehydrated through graded series of ethanol and rinsed in
water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase for 10 min.
Heat-induced antigen retrieval was performed in a microwave oven in 10 mM Tris/EGTA (pH =
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1069
9) by heating for 10 min followed by 15 min of boiling. Sections were cooled for 15 min at room
temperature and rinsed in Tris-buffered saline (TBS) with 0.05% Tween 20 for at least 5 min.
Sections to be incubated in anti TNF-alpha were additionally treated with 0.5% casein in TBS for
10 min to block non-specific binding sites. Section treated with polymer-HRP for 30 min
followed by visualization with DAB chromogen for 12 min. Sections were rinsed in water, and
0.5% cobber sulfate in TBS was added to enhance the staining intensity. Between incubations the
sections were washed several times in TBS buffer.
RESULTS
The result showed the activities of TBARS, SOD, CAT, GPx, and non enzymatic
antioxidants (GSH) levels in liver. Aluminium sulphate induced rats had shown decreased
activities of antioxidant enzymes when compared to control animals. Oral administration of
W.volubilis and kampferol to aluminium sulphate administered rats showed a significant
reduction in LPO levels and significant increase in the activities of antioxidant enzymes when
compared to aluminium sulphate induced rats. (Table.1)
Table : 1Effect of MEWV on LPO and antioxidant enzymes in Al2(SO4)3 induced rats
Parameters
(U/L)
Group I Group II Group III Group IV Group V
MDA 2.31±0.16 4.17±0.49* 3.84±0.21 a 4.05±0.27 a 2.27±0.19
GSH 47.6±1.9 15.5±0.11* 31.6±1.2 a 18.7 ± 1.1 a 46.5±3.2
SOD 7.41±0.15 3.3±0.18* 5.1±0.43 a 21.4 ± 1.7 a 7.39±0.68
CAT 6.6±1.5 3.2±0.16* 6.4±0.2 a 15.6± 1.2 a 7.3±0.41
GPx 48.6±2.5 19.8±1.4* 26.4±2.7 a 28.2 ± 1.6 a 47.3±0.41
Results are expressed as mean± S.E.M, n= 6 *P˂0.001, statistically significant as compared with
Al2(SO4)3 induced group. Values are expressed as SOD- mµ of epinephrine oxidized /min/mg
protein; GPx- mg of GSH reduced /g tissue; GSH- mg/g/ tissue.
Immunohistochemistry-TNF-alpha
Immuno-stained liver of control and MEWV groups for TNF – alpha expression showed
normal hepatic architecture. Liver of aluminium sulphate intoxicated group showed an increase
in the expression of TNF – alpha in the cytoplasm in the area of coagulative necrosis surrounding
hepatic central vein compared with that of negative control. The liver of the protective group
administered MEW plus aluminium sulphate showed no expression for TNF- α supporting the
protective effect of aluminium sulphate on hepatic toxicity (Figure 1).
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1070
Figure: 1 Immunohistochemical changes of
Tumor Necrosis Factor – alpha
Group I- Normal Control Group II- Al2 (SO4)3 (50 mg/kg/b.wt)
Group III- Al2(SO4)3 Group IV- Al2(SO4)3
+MEWV (200 mg/kg/b.wt) +Silymarin (25 mg/kg/b.wt)
Group V-MEWV (200 mg/kg/b.w)
MMP-2 protein expression by SDS PAGE
The expression of matrix metalloproteinase in control and experimental group of rat was
evaluated. A substantial increase in MMP was observed following treatment with Al2 (SO4)3
compared with control group of rats. No significant changes were observed in methanolic extract
of W.volubilis administered group of rats for a period of 30 days compared to that of control
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1071
group of rats. However, the expression of matrix metalloproteinase gradually decreased by the
methanolicextractof W.volubilis treatment as compared to the Al2(SO4)3 induced group of rats.
Similarly, silymarin treatment to Al2(SO4)3 induced mice showed significant reduction in
matrix metalloproteinase expression (Figure 2). So these results confirmed that W.volubilis
andsilymarinhave the ability to reduce the expression of matrix metalloproteinase during free
radical produced oxidative damage in liver tissues.
Fig : 2 MMP-2 protein expression by SDS PAGE
Lane M - Marker Lane
Lane 1 – Normal rats showed normal expression of MMP-2
Lane 2 –Group II liver tissue showed elevated expression of MMP-2
Lane 3 –Extract treated hepatotoxicity induced rats showed very mild expression of MMP-2
Lna e 4 – Silymarin treated hepatotoxicity induced rats down regulated the expression of MMP-2
Lane 5 –Extract treated rats showed normal expression of MMP-2.
DISCUSSION
Aluminium toxicity cause oxidative damage to the plant system by activating the
production of reactive oxygen species [33] . These ROS like superoxide radical (O2-), hydroxyl
radicals (OH-), singlet oxygen (1O2) and hydrogen peroxide (H2O2) generally are detoxified by
enzymatic antioxidant system. ROS if not detoxified causes serious damage to macro molecules
such as proteins, lipids and nucleic acids. In order to scavenge ROS and to combat oxidative
stress.
In vivo antioxidant defence mechanism fight againstfree radicals and reactive oxygen
species induceddamage, in which the endogenous enzymatic andnon-enzymatic antioxidants
such as glutathione, lipid peroxidase and superoxide dismutase [34] .Glutathione is as an
essential intracellular reducingsubstance for the maintenance of thiol groups onintracellular
M 1 2 3 4 5
Kda 94.0
66.2
71.8
26.0
33.0
MMP-2
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1072
proteins and antioxidant molecules inliving organisms. Perturbation ofglutathione status in a
biological system has beenreported to lead serious consequences [35]. Glutathione conjugatewith
free radicals directly, marking them ready forrenal excretion, which is especially important
fordealing with the products of hepatic cytochrome P 450 enzyme activity. The sulf-hydryl
portion of theglutathione can be used to reduce a variety of freeradicals in a reaction catalyzed by
the antioxidantenzyme, glutathione peroxidase [36]. Lipid peroxidase, superoxide dismutase
andother antioxidant enzymes constitute a mutuallysupportive team of defense against reactive
oxygenspecies. Superoxide dismutase is a metalloproteinaseto detoxify superoxide anions as an
efficientdismutative mechanism and is the first enzymeinvolved in the antioxidant defense[37].
However, once the balance between reactive oxygen species production and antioxidant
defensesis lost, oxidative stress consequently occurs, whichthrough a series of biological events
deregulates thecellular functions leading to various pathologicalconditions [38] .The results of
the treatment of kaempferol effectively blocked the aluminium sulphatecaused abnormal changes
in the level of glutathione,indicated that kaempferol has a potent antioxidantproperty towards
chemical induced hepatic injury[39]. Elevated level of MDA in aluminium sulphatetreated rats
indicates excessive formation of freeradicals and activation of lipid peroxidation systemresulting
in hepatic damage. The significant decline inthe concentration of TBARS in the rat’s liver tissues
of rats,treated with aluminium sulphate and MEWV indicates anti-lipid peroxidative effect of
watakaka volubilis.
In the present study, the activities of antioxidant enzymes like SOD, CAT and GPx, in rat
liver were dramatically decreased by the treatment of aluminium sulphate. This decrease could
be due to a feed back inhibition or oxidative inactivation of enzyme protein due to excess ROS
generation. The generation of H2 O2 may also lead to inactivation of this enzyme [40] .SOD
requires copper and zinc for its activity. The reduced activity of SOD in the presence of
aluminium sulphate may cause accumulation of O2∙-, H2O2 or the products of its
decomposition. The SOD activity was elevated in rats dosed with MEWV with aluminium
sulphate. This elevation may be due to the presence of antioxidant bioactive compound such as
phenolic compound which responsible for scavenging the super oxide anion radicals which was
evident by similar findings of [41]. Catalase is one of the important enzymes in the supportive
team of defense against reactive oxygen species (ROS). Catalase is a haemoprotein containing
four haem groups, that catalyses the decomposition of H2O2 to water and O2 and thus protects
the cell from oxidative damage by H2O2 and OH– [42].
Glutathione peroxide (GPx) has a well established role in protecting cells against
oxidative injury. GPx is a selenium containing metalloenzyme, partially located within cellular
membranes, which can remove hydrogen peroxide by converting reduced glutathione into
oxidized glutathione. GPx can also terminate the chain reaction of lipid peroxidation by
removing lipid hydroperoxides and H2O2 from the cell membrane. The decreased activity of
GPx in aluminium sulphate intoxicated group might be correlated to the decreased availability of
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1073
its substrate GSH. After the treatment with a MEWV improved the GPx levels significantly to
near normal [43].
Immunohistochemistry-TNF-α
TNF – α is a central pro-inflammatory cytokines. Activated kupffer cells produce
various mediators, including cytokines, proteases and oxygen radicals that participate in
inflammation, immune responses and modulation of hepatocyte metabolism.
Authors defend the use of molecular methods in clinical practice to perform differential
diagnosis; and to study biological targets for novel therapies [44]. Oxidative stress is one of the
responses for the production of pro -inflammatory cytokines, among which TNF-α, transforming
growth factors alpha (TGF-α) and beta (TGF-β), interleukins 6 (IL-6) and 8 (IL-8), nuclear
factor-kappa B (NFκB) and adiponectin stand out. These cytokines are produced by lymphocytes
and Kupffer cells, through free radical-mediated mechanisms, by altering mitochondrial
membrane permeability and inhibiting the respiratory chain [45,46].
Increased expression of TNF- α in aluminium sulphate rats may be due to inflammation,
necrosis and oxidative stress. Supplementation of Wattakakavolubilis effectively decreased TNF-
α expression in hepatic aluminium sulphate rats. Decreased TNF- α expression may be due to
attenuated inflammation, necrosis, and reduce the oxidative stress.
MMPs are important in many normal biological processes including embryonic
development, angiogenesis, and wound healing, as well as in pathological processes such as
inflammation, cancer and tissue destruction. The Lane 1 which consists of Normal rats with
normal expression of MMP-2. The Lane 2 which includes Group II liver tissue showed elevated
expression of MMP-2. The lane 3 Extract treated hepatotoxicity induced rats showed very mild
expression of MMP-2. The lane 4 Silymarin treated hepatotoxicity induced rats down regulated
the expression of MMP-2. From this study it cames to know that, MMP2 get altered after the
ingestion of Aluminium sulphate the MMP2 returns to normal level after herbal treatment. This
indicates the MMP2 involves in inflammation of liver injury.
CONCLUSION
It has been concluded that kaempferol is a tremendous bioactive flavanoid with potent
hepatoprotective activity. It proves the antioxidant enzymes and tumour necrosis factor.The
present study clearly reflected the effectiveness of the extract in ‘in vivo’ in terms of lipid
peroxidation inhibitory capacity and further confirmed the significant hepatoprotective activity
of its antioxidant mechanisms of action.
REFERENCES
1. Osinska E, Kanoniuk D, Kusiak A. Aluminium hemotoxicity mechanisms. Ann Univ
Mariae Curie Sklodowska [Med]. 2004; 59: 411-6.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1074
2. Williams RJP. What is wrong with aluminium? The J.D. Birchall Memorial Lecture. J
Inorg Biochem. 1999; 76: 81-8.
3. Yokel RA. Brain uptake retention and efflux of aluminum and manganese. Coordination.
Chem Rev. 2002; 228: 97-113.
4. Perl DP, Bondy AR. Alzheimer’s disease: X-ray spectrometric evidence of aluminium
accumulation in neuro fibrillary tangle bearing neurons. Science. 1980; 208: 297-9.
5. Droge, W. 2002. Free radicals in the physiological control of cell function. Physiol. Rev.
82: 47–95.
6. Halliwell, B, Gutteridge, J.M.C. 1999. Free radicals in biology and medicine, 3 rd
edition, Oxford University press, London, UK.
7. Haddad, J.J, Olver, R.E. & Land S.C. 2000. Antioxidant/prooxidant equilibrium
regulates HIF-1alpha and NF-kappa B redox sensitivity. Evidence for inhibition by
glutathione oxidation in alveolar epithelial cells. J. Biol. Chem. 275: 21130–21139.
8. Suzuki, S, Nakamura, S, Sakaguchi, T, Ochiai, H, Konno, H, Baba, S. & Baba S. 1997.
Alteration of reticuloendothelial phagocytic function and tumor necrosis factor-alpha
production after total hepatic ischemia. Transplantation 64: 821–827.
9. Flach, R, Speidel, N, Flohe, S, Borgermann, J, Dresen, I.G, Erhard J. & Schade F.U.
1998. Analysis of intragraft cytokine expression during early reperfusion after liver
transplantation using semi-quantitative RT-PCR. Cytokine 10: 445–451.
10. Zhong. J, Deaciuc, I.V, Burikhanov, R. & de Villiers, W.J. 2006. Lipopolysaccharide-
induced liver apoptosis is increased in interleukin-10 knockout mice. Biochim. Biophys.
Acta 1762: 468–477.
11. Schwabe, R.F. & Brenner D.A. 2006. Mechanisms of liver injury. I. TNF-alpha-induced
liver injury: role of IKK, JNK, and ROS pathways. Am. J. Physiol. Gastrointest. Liver
Physiol. 290: G583–G589.
12. Oyaizu M., studies on product of browning reaction prepared from glucose amine. J.
Nutr., 1986; 44: 307-315.
13. Halliwell B. and Gutteridge J.M.C., Freeradicals, other reactive species and
disease; Clarendon Press, Oxford, 1989.
14. Kinsella J.E., Frankel E., German B. and Kanner J., Possible mechanisms for the
protective role of antioxidants in wineand plant foods, Food Technol., 1993; 47:85-89.
15. Good PF, Perl DP, Bierer LM, Schmeidler J. Selective accumulation of aluminum and
iron in the neurofibrillary tangles of Alzheimer’s disease: a laser microprobe (LAMMA)
study. Ann Neurol 1992; 31: 286-92.
16. Bjertness E, Candy JM, Torvik A, Ince P, McArthur F, Taylor GA. Content of brain
aluminum is not elevated in Alzheimer disease. Alzheimer Dis Assoc Disord, 1996 ;10:
171-4.
17. Flaten TP.Aluminium as a risk factor in Alzheimer’s disease, with emphasis on drinking
water. Brain Res Bull. 2001; 55: 187-96.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1075
18. Hirsch EC, Brandel JP, Galle P, Javoyagid F, Agid Y. Iron and aluminum increase in the
substantia nigra of patients with Parkinson’s disease: an x-ray microanalysis. J
Neurochem , 1991 ; 56: 446-51.
19. Erasmus RT, Kusnir J, Stevenson WC, Lobo P, Herman MM, Wills MR.
Hyperalbuminemia associated with liver transplantation and acute renal failure. Clin
Transplant. 1995; 9: 307-11.
20. Yumoto S, Ohashi H, Nagai HS, Kakimi A, Ishikawa K, Kobayashi. Aluminum toxicity in
the rat liver and brain. Nucl Instrum Methods Phys Res B. 1993;75: 188-90.
21. Chinoy, NJ, Patel TN. Reversible toxicity of fluoride and aluminium in liver and
gastrocnemius muscle of female mice. Fluoride. 1999; 32: 215-29.
22. Moumen R, Ait-Oukhatar N, Bureau F, Fleury C, Bougle D, Arhan P. Aluminium
increases xanthine oxidase activity and disturbs antioxidant status in the rat. J Trace
Elem Med Biol. 2001; 15: 89-93.
23. Anane R, Creppy EE. Lipid peroxidation as a pathway to aluminium cytotoxicity in
human skin fibroblast cultures: prevention by superoxide dismutase plus catalase and
vitamins E and C. Hum Exp Toxicol. 2001; 20: 477-81.
24. Missel JR, Schetinger MR, Gioda CR, Bohrer DN, Packolski IL, Zanatta N. Chelating
effects of novel pyrimidines in a model of aluminum intoxication. J Inorg Biochem. 2005;
99: 1853-57.
25. Koperuncholan M and Ahmed John S. Biosynthesis of Silver and Gold Nanoparticles and
Antimicrobial Studies of Some Ethno medicinal Plants in South-Eastern Slope of Western
Ghats, IJPI’s- Journal of Pharmacognosy and Herbal Formulations. 2011; 1: 10-5.
26. Rajadurai M, Vidhya VG, Ramya M and Bhaskar A . Ethno-Medicinal plants used by the
Traditional Healers of Pacchamalai Hills, Tamil Nadu, India. Ethnomedicine. 2009; 3:
39-41.
27. Muthu C, Ayyanar M, Raja N and Ignacimuthu S. Medicinal plants used by traditional
healers in Kancheepuram District of Tamil Nadu, India. Journal of Ethnobiology and
ethnomedicine. 2006; 2: 43-52.
28. Pandikumar P, Ayyanar M and Ignacimuthu S. Medicinal plants used by Malasar tribes
of Coimbatore district, Tamil Nadu. Indian Journal of Traditional Knowledge. 2007; 6:
579-82.
29. Ayyanar M, Sankara Sivaraman K and Ignacimuthu S. Traditional Herbal Medicines
used for the treatment of Diabetes among two major tribal groups in South Tamil Nadu,
India.Ethno Botanical Leaflet. 2008; 47: 389-394.
30. Fraga CG, Oteiza PI, Golub MS, Gershwin ME, Keen CL. Effects of aluminum on brain
lipid peroxidation. Toxicol Lett. 1990; 51: 213-19.
31. Kakkar P, Das B, Viswanathan PN. Indian J Biochem Biophys. 1984; 2: 130–2
32. Ellman GL. Tissue sulfhydryl Groups: Archives of Biochemistry and Biophysics. 82,
33. Sinha KA. Colorimetric Assay of Catalase. Analytical Biochemistry. 1972; 47: 389-94.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1076
34. Mohandas J, Marshall JJ, Duggin, GG, Horvath JS, Tiller D. Differential distribution of
glutathione and glutathione related enzymes in rabbit kidneys: possible implication in
analgesic neuropathy. Cancer Research. 1984; 44: 5086–91.
35. Shah K, Kumar RG, Verma S, Dubey RS. Effect of cadmium on lipid peroxidation,
superoxide anion generation and activities of antioxidant enzymes in growing rice
seedlings. Plant Science. 2001; 161: 1135–44.
36. Chow CK. Nutritional influence on cellularantioxidant defence systems. American J.
ofClinical Nutrition. 1979; 32: 1066–81.
37. Bandyopadhyay U, Das D and Banerjee RK. Reactiveoxygen species: Oxidative damage
and pathogenesis.Current Science. 1999; 77: 658–66.
38. Webb C and Twedt D. Oxidative stress and liverdisease. Veterinary Clinics of North
America: SmallAnimal Practice. 2008; 38: 125–135.
39. Salvemini D, Riley DP and Cuzzocrea S. SOD mimetic are coming of age. Nature Review
DrugDiscovery. 2002; 367–74.
40. Sahu SC and Gray GC. Pro-oxidant activity offlavonoids: effects on glutathione and
glutathione S-transferase in isolated rat liver nuclei. Cancer Letters. 1996; 104: 193–6.
41. Chen CJ, Deng AJ, Liu C, Shi R, Qin HL and WangAP. Hepatoprotective activity of
Cichorium endivia L.extract and its chemical constituents. Molecules. 2011; 16:9049–66.
42. Ohata H, Kitauchi S, Yoshimura N, Mugitani K, Iwane M. Progression of chronic
atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric
cancer. J. Cancer. 2004; 109: 138-43.
43. Cao G, Alessio HM, and Cutler RG. Oxygen-radical absorbance capacity assay for
antioxidants. Free Radical Biol. Med. 1993; 14: 303–11.
44. Tolbert NE. Metabolic pathways in peroxisomes and glyoxysomes. Annual Review of
Biochemistry. 1981; 50: 133–57.
45. Roberta JW, Timothy JP. Free radicals. Clinical bio-chemistry metabolic and clinical
aspects, edited by Williams J. Marshall, 1995; 765-77.
46. Kang, M, Oh, J.W, Lee, H.K, Chung, H.S, Lee, S.M, Kim, C, Lee, H.J, Yoon, D.W, Choi
H, Kim H, Shin M, Hong, M, Bae H. 2004. Anti-obesity effect of PM-F2-OB, an anti-
obesity herbal formulation, on rats fed a high-fat diet. Biol Pharm Bull, 27(8): 1251-
1256.
47. Paolo Zatta, T.K, Mario Suwalsky, Guy Berthon, 2002. Aluminum (III) as a promoter of
cellular oxidation, Coord. Chem. Rev. 228: 271–284.
48. Brahmachari, G. 2009. Mother nature: An inexhaustible source of drugs and lead
molecules. In Brahmachari G, editor. Chemistry, Biochemistry and Pharmacology (1st
edn). Narosa Publishing House Pvt. Ltd: New Delhi. 1-20.
Journal of Information and Computational Science
Volume 10 Issue 1 - 2020
ISSN: 1548-7741
www.joics.org1077